We are developing a live-attenuated, intranasal, pediatric vaccine against human respiratory syncytial virus (HRSV). Building on years of molecular and biologic studies, we use reverse genetics to produce highly defined vaccine candidates from cloned cDNAs. Five cDNA-derived viruses have been evaluated to date in clinical studies, as described in previous years. The most promising virus, called rA2cp248/404/1030/delSH, was safe and immunogenic in young infants. However, there was evidence of loss of the 248 or 1030 attenuating point mutation (Tyr-1321-Asn or Gln-831-Leu, respectively, in the L protein) in some isolates. This instability is due to the high mutation rate of the virus (10-4) and the fact that the amino acid point mutations each involve only a single nt substitution and can thus readily revert. By examining all 20 amino acid assignments at a given site, one can segregate them into attenuating versus wild-type-like, and can examine the genetic code to find an attenuating codon that differs by 2 or, preferably, 3 nt from any wild-type-like codon. Reversions needing 2 or 3 nt substitutions would be exponentially less frequent (10-8 and 10-12, respectively). It is not always possible to obtain a 2- or 3-nt difference, but even then one often can find a codon that, while still susceptible to a single nt reversion, has fewer such possibilities than other codons. This seems to provide a more modest increase in stability. Last year, we described such an analysis for the 248 mutation. Such a study is laborious, since one must make 20 viruses, sequence each completely to identify possible adventitious mutations, remake 3 to 5 viruses due to such mutations, characterize phenotypes in vitro and in mice, and perform stress test stability studies by passage in vitro under increasing non-permissive temperature to monitor reversion (involving phenotypic and sequence analysis of passaged viruses). For the 248 mutation, we were not able to identify an appropriate codon with a 2- or 3-nt difference, but do have evidence that a particular codon choice provides a more modest increase in stability. During the present year, we have extended this analysis to the 1030 mutation, and have nearly completed the study. In other work, we successfully prepared clinical lots of (i) wild type HRSV, for use in pathogenesis studies as well as challenge virus in studies by others, and (ii) two versions of a mutant in which the M2-2 protein has been deleted as a vaccine candidate. The M2-2 protein appears to function in regulating RNA synthesis, since its deletion results in a decrease in RNA replication and an increase in gene transcription and antigen synthesis. HRSV readily reinfects during infancy and throughout life without the need for significant antigenic change. This is frequently interpreted as evidence that HRSV inhibits or subverts protective immunity. In last years report we showed that re-infection may be aided by a viral immune evasion mechanism involving a secreted form of the attachment G protein. As another approach to this issue, we also initiated study of the effects of HRSV and other respiratory viruses on human dendritic cells (DC) in vitro. These are potent antigen presenting cells that play a major role in initiating and modulating the adaptive immune response. We compared the effects of HRSV on human monocyte-derived DC (MDDC) in a side-by-side comparison with HMPV and HPIV3 using GFP-expressing viruses. We also included influenza A virus (Flu) in the comparison, since Flu induces a strong immune response and rarely re-infects symptomatically without antigenic change. The three human paramyxoviruses infected DCs poorly, whereas a Newcastle disease virus-GFP control infected very efficiently and Flu also is known to infect relatively efficiently. With HRSV, HMPV, and HPIV3, only a few percent of cells were robustly infected (i.e., GFP+), with the remainder having low, abortive levels of viral RNA synthesis. Infection at the individual cell level tended to be relatively benign, such that GFP+ cells were neither more nor less able to mature compared to GFP- bystanders. Low infectivity and low intracellular antigen synthesis by HRSV, HMPV, and HPIV3 likely would reduce the ability of these DC to activate of CD8+ T cells important for host defense, which is presently being investigated. All four viruses induced low-to-moderate maturation of DC and cytokine/chemokine responses, and thus HRSV was not distinct from Flu on this basis. We analyzed MDDC that were exposed to these viruses in vitro for the expression of 62 genes pertinent to maturation. One of these was CCR7, which normally is up-regulated during maturation and directs migration of antigen-bearing DC to T cell-rich zones in lymphatic tissue. We found that DCs infected with HRSV or HMPV did not efficiently up-regulate CCR7, in contrast to HPIV3 and, especially, Flu. This was confirmed at the level of cell surface protein expression. In addition, HMPV and HRSV did not efficiently down-regulate surface expression of CCR1, 2 and 5, which maintain DC residence in peripheral tissues and normally are down-regulated during maturation. In an in vitro migration assay, HRSV- and HMPV-treated DC migrated less efficiently to the CCR7 ligand CCL19, which directs DC chemotaxis to lymphatic tissue. Secondary stimulation with lipopolysaccharide reversed this phenotype, suggesting that it is due to suboptimal stimulation rather than irreversible inhibition. This phenotype appeared to be partly due to reduced expression of pro-inflammatory cytokines by DC treated with HMPV and HRSV. Inefficient migration of DC in response to HRSV and HMPV infection could contribute to a dampening of the adaptive response to these viruses. We investigated whether these viruses differentially affect interactions between dendritic cells (DC) and CD4 T cells, as has been widely hypothesized. We infected human MDDC with HRSV, HMPV, HPIV3, or Flu, and compared their ability to induce proliferation of autologous CD4+ T cells in vitro. We investigated both virus-specific memory responses as well as superantigen-induced responses. In general, there was little evidence of virus-specific inhibition. There was a trend of increasing memory responses, HMPV <HRSV <HPIV3 <Flu, but the differences were not significant. Overall, cytokine production by the proliferating T cells was similar among the different viruses, with no evidence of Th2 or Th17 skewing. These results provided no evidence of marked differences between the viruses in their effects on CD4 T cell activation. We recently demonstrated that the HRSV NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, also has a suppressive effect on the maturation of human dendritic cells (DC) due in part to suppression of IFN-I production. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with HRSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by flow cytometry. Deletion of the NS1, but not NS2, protein (i) increased the proliferation and activation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to the respiratory mucosa and triggers cytolytic activity (ii) increased the activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects, and (iii) skewed the Th1/Th2 balance towards Th1 by reducing the number of IL-4-producing CD4+ T cells, which are associated with enhanced RSV disease. Taken together, these data demonstrate that expression of NS1 by wild type HRSV suppresses two protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes Th2 cells that can enhance HRSV disease.